Ever since the discovery of Ras in 1981, a number of small GTP binding proteins (small G proteins) similar to Ras have been found, and many physiological functions they possess have been studied. These small. G proteins have a molecular weight of 20,000-30,000 and do not have a subunit structure. They all specifically bind GDP and GTP, and hydrolyze the thus-bound GTP (GTPase activity) (Hall, A., Science, 249, 635-640, 1990; Bourne, H. R. et al., Nature, 349, 117-127, 1991).
To date, more than 50 kinds of genes encoding these small G proteins have been found from yeast to mammals, forming a superfamily. These small G proteins are largely divided into 5 groups of Ras, Rho, Rab, Arf and others, according to the similarity of their amino acid sequences.
Of these, Rho was named so because its gene isolated in the form of cDNA from sea hare neuromuscle encodes a polypeptide having about 35% homology with Ras (Ras homologue) (Madaule, P., Cell, 41, 31-40, 1985).
Rho is specifically ADP ribosylated by C3 enzyme, which is one of the botulinum toxins, and Staphylococcal toxin EDIN, and inactivated (Narumiya, S. and Morii, S., Cell Signal, 5, 9-19, 1993; Sekine, A. et al., J. Biol. Chem., 264, 8602-8605, 1989). Hence, the C3 enzyme and EDIN were used to study the involvement of Rho in cell functions from various aspects.
For example, phosphorylation by myosin light chain (MLC) kinase is considered to enable actin-myosin interaction and initiate contraction of smooth muscle, and the structure of smooth muscle myosin phosphatase, which dephosphorylates MLC, has been clarified (Shimizu, H. et al., J. Biol. Chem., 269, 30407-30411). It has been clarified that the activity of myosin phosphatase is, like MLC kinase, under the control of the intracellular signal transduction system and Rho is involved in this mechanism. Moreover, an active Rho bound with GTP has been found to enhance Ca-dependent contraction in a smooth muscle skinned fiber specimen (Hirata, K., J. Biol. Chem., 267, 8719-8722, 1992), thereby suggesting that the increase in Ca sensitivity in smooth muscle contraction is caused by the inhibition of myosin phosphatase activity via Rho.
In Swiss 3T3 cell and 3Y1 cell, moreover, Rho-dependent promotion of tyrosine phosphorylation (Kumagai, N. et al., J. Biol. Chem., 270, 8466-8473, 1993) and activation of many kinds of serine/threonine kinases (Kumagai, N. et al., FEBS Lett., 366, 11-16, 1995) have been acknowledged. From this, the presence of plural protein kinases in the downstream of Rho in the signal transduction pathway via Rho has been suggested and, serine/threonine kinase (Rho kinase) activated along with the activation of Rho, such as ROCα (Leung, T. et al., J. Biol. Chem., 270, 29051-29054, 1995) [another name Rho-kinase, ROCK-II] and p160ROCK (Ishizaki, T. et al., EMBO J., 15, 1885-1893, 1996) [another name ROCKβ, ROCK-I], have been reported as one of the proteins that transduct signals from Rho.
In addition, it has been reported that this Rho kinase directly phosphorylates myosin phosphatase and inhibits its activity (Kimura, K. et al., Science, 273, 245-248, 1996).
In recent years, a certain kind of amide compound has been found to be a selective Rho kinase inhibitor (Uehata, M. et al., Nature, 389, 990-994, 1996, WO 98/06433), and further, certain kinds of isoquinoline sulfonamide derivatives (WO 98/06433) and isoquinoline derivatives (Naunyn-Schmiedeberg'S Archives of Pharmacology 385(1) Suppl. R219 1998, 11) have been found to be Rho kinase inhibitors.
In addition, certain kinds of vinylbenzene derivatives such as ethacrynic acid, 4-[2-(2,3,4,5,6-pentafluorophenyl)-acryloyl]cinnamic acid and the like, and cinnamic acid derivatives have been recently reported as Rho kinase inhibitors (WO 00/57914, JP-A-2000-44513).
Particularly, various physiological functions of signal transduction via Rho-Rho kinase have been elucidated using (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane, which is one of the above-mentioned selective Rho kinase inhibitors.
For example, it has been clarified that a selective Rho kinase inhibitor suppresses formation of desmosome and a stress fiber, which is due to the stimulation of Rho or LPA (lysophoshatidic acid), and shows an inhibitory activity on the contraction caused by calcium sensitivity accentuation in smooth muscle (Uehata, M. et al., Nature, 389, 990-994, 1996).
In addition, this inhibitor has been reported to be involved in various cell functions such as an inhibitory action on neurite degeneracy by LPA in nerve cell-derived cultured cell, NIE-115 cell (Hirose, M. et al., J. Cell Biol. 141, 1625-1636, 1998), an inhibitory action on the activation of 1 type Na+—H+ exchanger (Tominaga, T. et al., EMBO J. 17, 4712-4722, 1998).
Furthermore, a concentration-dependent inhibition of AH cell invasion by a specific ROCK/Rho kinase inhibitor in an invasion model of rat ascites hepatoma (AH cell) into homogeneous rat single layer mesothelial cell layer has been reported (Itoh, K. et al., Nature Med. 5, 221-225, 1999), that kinematic accentuation of cells via Rho-Rho kinase has been found to be critical to cancer cell invasion and metastasis, and further that transformation via Rho-Rho kinase is critical in the malignant alteration of cancer cells (Sahai, E. et al., Curr. Biol. 9, 136-145, 1999).
A signal transduction via Rho-Rho kinase is considered to be involved in a great diversity of cell phenomena, such as smooth muscle contraction, cell movement, cell adhesion, morphological change of cells, cell growth and the like, and therefore, a drug that blocks the function of Rho-Rho kinase has a potential of becoming a therapeutic agent of diseases such as hypertension, pulmonary hypertension, angina pectoris, cerebrovascular contraction, asthma, peripheral circulation disorder, glaucoma, erectile dysfunction and the like, in which smooth muscle contraction is involved, invasion and metastasis of cancer, angiostenosis, arteriosclerosis, retinopathy, immune response, fibrosing disease, ischemia-reperfusion injury and the like, in which cell movement is involved, metastasis of cancer, inflammation, autoimmune disease, AIDS, ischemia-reperfusion injury and the like, in which cell adhesion is involved, brain function disorder, osteoporosis (bone formation and resorption) and the like, in which morphological change of cell is involved, and cancer, arteriosclerosis, ischemia-reperfusion injury and the like, in which cell growth is involved.
Accordingly, a specific Rho kinase inhibitor can be a therapeutic drug of various diseases, and creation of a superior new compound is desired.
It is an object of the present invention to provide a novel compound having a Rho kinase inhibitory activity, which has a potential of becoming a therapeutic drug of the diseases in which Rho-Rho kinase is involved.
The present inventors have conducted intensive studies in view of the above-mentioned situation, and found that a novel amide compound represented by the following formula (I), an isomer thereof and a pharmaceutically acceptable salt thereof have a potent Rho kinase inhibitory activity and completed the present invention. They have also found that the compound of the present invention can be useful as a therapeutic agent for various diseases where Rho-Rho kinase is involved, a reagent having a Rho kinase inhibitory activity and a diagnostic agent of diseases caused by Rho kinase, which resulted in the completion of the present invention.